P16 expression constructs and their application in cancer therapy

ABSTRACT

A variety of genetic constructs are disclosed that will find both in vitro and in vivo use in the area of tumor biology and cancer therapy. In particular, expression constructs are provided that contain a p16 encoding region and other regulatory elements necessary for the expression of a p16 transcript. One version of the expression construct is a replication-deficient adenoviral vector. Also provided are methods for the transformation of cell lines and the inhibition of cancer cell proliferation.

This is a continuation of application Ser. No. 08/502,881 filed Jul. 17,1995.

The government may own certain rights in the present invention pursuantto grant number CA 45187 from the National Institutes of Health and CoreGrant CA 16672.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of tumor biology.In particular, the invention relates to a nucleic acid encoding a tumorsuppressor and its use in inhibiting tumor growth. In one embodiment,the invention relates to expression constructs encoding p16 and theiruse in inhibiting cancer.

2. Description of the Related Art

Cancer is one of the leading causes of human disease, being responsiblefor 526,000 deaths in the United States each year (Boring et al., 1993).Lung cancer alone kills more than 140,000 people annually in the UnitedStates. Recently, age-adjusted mortality from lung cancer has surpassedthat from breast cancer in women. Although implementation ofsmoking-reduction programs has decreased the prevalence of smoking, lungcancer mortality rates will remain high well into the twenty-firstcentury. Unfortunately, current treatment methods for cancer, includingradiation therapy, surgery and chemotherapy, are known to have limitedeffectiveness. The rational development of new therapies for lung cancerlargely will depend on gaining an improved understanding of the biologyof cancer at the molecular level.

With advances in molecular genetics and biology, it has become evidentthat altered expression of normal genes can lead to the initiation oftransforming events that result in the creation of cancer cells. Theconventional therapy for malignancy, such as chemotherapy and radiation,has focused on mass cell killing without specific targeting, oftenresulting in damaging side effects. A new direction in cancer therapy isto deliver a normal gene to replace or correct the mutated gene, therebyaltering the malignant phenotype of transformed cells. Severalexpression constructs have been developed in order to deliver a geneinto somatic cells with high efficiency.

Cells are regulated in both positive (stimulatory) and negative(suppressive) manners. Loss of negative regulation of cell growth isoften found in malignant cells. Accumulating molecular genetic evidencehas revealed that loss of negative regulators, or increase in positiveregulators in normal cells, can produce such cellular growthabnormalities. Most negative regulators (Marx, 1993; Grunicke and Maly,1993), referred to as tumor suppressors, have been found to be involvedeither in direct control of the cell cycle (e.g., Rb, p53, WT-1) or inthe signaling pathway leading to cell growth and differentiation (e.g.,NF-1). In addition, recent data suggest that genes related to themaintenance of cell architecture and polarity also may function as tumorsuppressors (Marx, 1993; Fearon et al., 1990; Trofatter et al., 1993).

The major transitions of the eukaryotic cell cycle are triggered bycyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4(CDK4), regulates progression through the G₁. The activity of thisenzyme may be to phosphorylate Rb at late G₁. The activity of CDK4 iscontrolled by an activating subunit, D-type cyclin, and by an inhibitorysubunit, the p16^(INK4) protein. p16^(INK4) has been biochemicallycharacterized as a protein that specifically binds to and inhibits CDK4,and thus may regulate Rb phosphorylation (Serrano et al., 1993; Serranoet al., 1995). Since the p16^(INK4) protein is a CDK4 inhibitor(Serrano, 1993), deletion of this gene may increase the activity ofCDK4, resulting in hyperphosphorylation of the Rb protein. p16 also isknown to regulate the function of CDK6.

p16^(INK4) belongs to a newly described class of CDK-inhibitory proteinsthat also includes p15^(B), p21^(WAF1), and p27^(KIP1). The p16^(INK4)gene maps to 9p21, a chromosome region frequently deleted in many tumortypes. Homozygous deletions and mutations of the p16^(INK4) gene arefrequent in human tumor cell lines. This evidence suggests that thep16^(INK4) gene is a tumor suppressor gene. This interpretation has beenchallenged, however, by the observation that the frequency of thep16^(INK4) gene alterations is much lower in primary uncultured tumorsthan in cultured cell lines (Caldas et al., 1994; Cheng et al., 1994;Hussussian et al., 1994; Kamb et al., 1994; Kamb et al., 1994; Mori etal., 1994; Okamoto et al., 1994; Nobori et al., 1995; Orlow et al.,1994; Arap et al., 1995). Restoration of wild-type p16^(INK4) functionby transfection with a plasmid expression vector reduced colonyformation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).

SUMMARY OF THE INVENTION

The present invention addresses the need for improved therapy for lungand other p16-associated cancers by providing expression constructscontaining a nucleic acid encoding p16. It also is an object of thepresent invention to provide methods for the use of such compositionsand, in particular, use in the treatment of cancer. In anotherembodiment, the present invention encompasses methods for transformingcells using a p16 nucleic acid in an expression construct.

The present invention also encompasses expression constructs thatcomprise a promoter functional in eukaryotic cells and a nucleic acidencoding p16, the nucleic acid being under transcriptional control ofthe promoter.

In a preferred embodiment, the expression constructs further comprise apolyadenylation signal. In another embodiment, the constructs furthercomprise a selectable marker. In a further embodiment, the expressionconstruct is an adenovirus. In a preferred embodiment, the expressionconstruct is an adenovirus that lacks at least a portion of the E1region.

In certain embodiments, the nucleic acid is a cDNA. In other embodimentsthe nucleic acid is a genomic DNA. Still other embodiments include acombination of cDNA and genomic DNA, for example, in a mini-geneconstruct. In an exemplary embodiment the nucleic acid is positioned ina sense orientation with respect to said promoter. In another embodimentthe nucleic acid is positioned in an antisense orientation.

The present invention also includes pharmaceutical compositionscomprising an expression construct with a promoter functional ineukaryotic cells and a nucleic acid encoding p16, along with apharmaceutically acceptable buffer, solvent or diluent. In certainembodiments, the expression construct and pharmaceutically acceptablebuffer, solvent or diluent are supplied in a kit.

The invention also provides a method for restoring proper p16 functionin a cell that lacks p16 function or expresses a functional p16 that isimproperly compartmentalized. This method comprises contacting such acell with an expression construct as described above, wherein thenucleic acid is positioned in a sense orientation. In an exemplaryembodiment of the invention, the cell is a transformed cell and thecontacting reverses the transformed phenotype. In a further embodiment,the cell is a lung, bladder, leukemia or melanoma cancer cell and, instill a further embodiment, the expression construct is an adenovirus.

The present invention further comprises a method for inhibiting p16function in a cell. This method comprises contacting such a cell with anexpression construct as described above, wherein the nucleic acid ispositioned in an antisense orientation. In a further embodiment, theexpression construct is an adenovirus.

Another embodiment of the invention is a method of treating a mammalwith cancer. This method comprises administering to an animal apharmaceutical composition comprising an expression construct having apromoter functional in eukaryotic cells and a nucleic acid encoding p16,positioned in a sense orientation, in a pharmaceutically acceptablebuffer, solvent or diluent. In a particular embodiment of the invention,the mammal is a human. In another embodiment, administering is viaintravenous injection. In a further embodiment, the cancer is lungcancer.

In further embodiments the present invention encompasses methods fordetecting cancer cells in a sample by detecting p16 or a nucleic acidencoding a p16.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A—Nucleotide Sequence of p16. (SEQ ID NO:1)

FIG. 1B—Amino Acid Sequence of p16. (SEQ ID NO:2)

FIG. 2—Cell growth curves of the Ad-p16 infected cell lines. Cells wereinoculated at densities of 5×10⁴ in 60 mm culture dishes 24 h beforeinfection and infected with Ad-p16 or Ad5CMV-lacZ at 50 PFU/cell.Culture medium alone was used for mock infection. Triplet cultures ofeach cell line for each treatment were counted daily from postinfectionday 1 to day 6. The curves are plotted from a representative assay ofthree experiments (Mean±SD).

FIG. 3—Tumor growth in mice following intratumoral injections of Ad-p16,control virus AD5CMV-lacZ or PBS (5 mice per group). Subcutaneous tumornodules were created by injecting 5×10⁶ H460 cells suspended in 0.1 mlof PBS into the dorsal flanks of nude mice. Tumor nodules (180 to 220mm³) were treated 16 days after cell implantation. Direct intratumoralinjection of Ad-p16, Ad5CMV-lacZ, or PBS was performed. For each tumornodule, 10¹⁰ PFU of Ad-p16 or Ad5CMV-lacZ divided equally in three doseswas injected on alternate days for 6 days. PBS injection served as acontrol. Tumor size was measured with linear calipers in two orthogonaldirections on alternate days after injection and tumor volumes werecalculated (Mean±SD).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Evidence now has accumulated that dysregulation of certain genesinvolved in control of the cell cycle contributes to the malignantprogression of cells. For example, premature entry of a cell into thenext phase of the cell cycle may result in incomplete repair of DNAdamage and subsequent genomic instability. As mentioned above,cyclin-dependent kinases play an important role in cell cycleregulation. It is known that p16^(INK4) protein can complex with cyclinD1-CDK4 and inhibit its interaction with Rb, thus retarding passagethrough the cell cycle. The presence of point mutations and homozygousdeletions in a high percentage of cancer cell lines further suggeststhat p16^(INK4) may function as a tumor suppressor gene.

Mutations in primary human esophageal and pancreatic cancers, bladdercancers, melanoma and NSCLC metastases have been reported, although therates of mutations in primary tumors are lower than those for cell lines(Mori et al., 1994; Okamoto et al., 1994; Okamoto et al., 1995; Zhou etal., 1994; Cairns et al., 1994; Hussussian et al., 1994; Kamb et al.,1994; Gruis et al., 1995). Deletions and mutations may not be theprimary mode of p16^(INK4) inactivation, however, as hypermethylation ofp16^(INK4) associated with transcriptional silencing is a frequentfinding in lung cancer, head and neck cancer, glioma cell lines andfresh tumors without deletions or mutations (Herman et al., 1995).

The data presented here are the first to show that p16 acts as a tumorsuppressor in vivo. Thus, the present invention addresses the need forimproved therapy for lung cancer and other p16-associated diseases. Inparticular, an expression construct capable of expressing a functionalp16 product can be used to inhibit tumor cell proliferation. Inaddition, the present invention encompasses the use of antisensemethodology, directed at p16, to transform cell lines or otherwiseincrease the rate or extent of growth of cells. The followingdescription provides a more detailed explanation of these and otheraspects of the present invention.

There also is evidence that p16-targeted treatments will havetherapeutic implications in an anti-angiogenic approach. There are manydisease where a decrease in vasculature is desirable. In addition,p16-treatments also may prove beneficial with respect tohyperproliferative disorders such as restenosis.

A. p16 AND p16-RELATED NUCLEIC ACIDS

The nucleic acid according to the present invention may encode an entirep16 gene, a functional p16 protein domain, or any p16 polypeptide,peptide or fragment that is sufficient to effect inhibition of CDK4. Thep16 nucleic acid may be derived from genomic DNA, i.e., cloned directlyfrom the genome of a particular organism. In preferred embodiments,however, the nucleic acid encoding p16 would comprise complementary DNA(cDNA) or cDNA plus an intron, i.e., a mini-gene.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA does not contain any non-coding sequencesbut, rather, contains only the coding region of the correspondingprotein. There may be times when the full or partial genomic sequence ispreferred, such as where the non-coding regions are required for optimalexpression or where non-coding regions such as introns are to betargeted in an antisense strategy.

Throughout the application, the term “p16” is used synonymously with itsother designations—MTS1, CDK4I and CDKN2. The use of the term “p16” alsois intended to refer to all p16 homologues from other species inaddition to those specified.

It also is contemplated that a given p16 may be represented by naturalvariants that have slightly different primary sequences but,nonetheless, are biological functional equivalents of each other (seebelow). In order to function according to the present invention, allthat is required is that the p16 bind to CDK4. To test for such anaffect, it is a simple matter to assay binding of a protein, encoded bya p16 nucleic acid, in vitro or by the use of transfection techniques asdescribed by Serrano et al., 1993, incorporated herein by reference.

As used in this application, the term “nucleic acid encoding a p16”refers to a nucleic acid molecule that has been isolated free of totalcellular nucleic acid. In preferred embodiments, the invention concernsa nucleic acid sequence essentially as set forth in FIG. 1A (SEQ IDNO:1). The term “as set forth in FIG. 1A” means that the nucleic acidsequence substantially corresponds to a portion of FIG. 1A and hasrelatively few codons that are not identical, or functionallyequivalent, to the codons of FIG. 1A. The term “functionally equivalentcodon” is used herein to refer to codons that encode the same aminoacid, such as the six codons for arginine or serine, and also refers tocodons that encode biologically equivalent amino acids (as in Table 1below).

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalnine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His HCAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu LUUA UUG CUA CUC CUG CU                     U Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CG                     USerine Ser S AGC AGU UCA UCC UCG UC                     U Threonine ThrT ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

Allowing for the degeneracy of the genetic code, sequences that havebetween about 50% and about 75%; or more preferably, between about 76%and about 99% of nucleotides that are identical to the nucleotides ofFIG. 1A will be sequences that are “as set forth in FIG. 1A.” Sequencesthat are essentially the same as those set forth in FIG. 1A may also befunctionally defined as sequences that are capable of hybridizing to anucleic acid segment containing the complement of FIG. 1A under standardconditions.

Suitable hybridization conditions will be well known to those of skillin the art. In certain applications, for example, substitution of aminoacids by site-directed mutagenesis, it is appreciated that lowerstringency conditions are required. Under these conditions,hybridization may occur even though the sequences of probe and targetstrand are not perfectly complementary, but are mismatched at one ormore positions. Conditions may be rendered less stringent by increasingsalt concentration and decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15M to about 0.9M salt, attemperatures ranging from about 20° C. to about 55° C. Thus,hybridization conditions can be readily manipulated, and thus willgenerally be a method of choice depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 μM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C. Formamideand SDS also may be used to alter the hybridization conditions.

Naturally, the present invention also encompasses DNA segments that arecomplementary, or essentially complementary, to the sequence set forthin FIG. 1A. Nucleic acid sequences that are “complementary” are thosethat are capable of base-pairing according to the standard Watson-Crickcomplementary rules. As used herein, the term “complementary sequences”means nucleic acid sequences that are substantially complementary, asmay be assessed by the same nucleotide comparison set forth above, or asdefined as being capable of hybridizing to the nucleic acid segment ofFIG. 1A under relatively stringent conditions such as those describedherein. Such sequences may encode the entire p16 molecule or functionalfragments thereof.

Alternatively, the hybridizing segments may be shorter oligonucleotides.Sequences of 17 bases long should occur only once in the human genomeand, therefore, suffice to specify a unique target sequence. Althoughshorter oligomers are easier to make and increase in vivo accessibility,numerous other factors are involved in determining the specificity ofhybridization. Both binding affinity and sequence specificity of anoligonucleotide to its complementary target increases with increasinglength. It is contemplated that oligonucleotides of 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or more base pairs will be used. Sucholigonucleotides will find use, for example, as probes and as primers inamplification reactions.

The DNA segments of the present invention include those encodingbiologically functional equivalent p16 proteins and peptides. Suchsequences may arise as a consequence of codon redundancy and functionalequivalency that are known to occur naturally within nucleic acidsequences and the proteins thus encoded. Alternatively, functionallyequivalent proteins or peptides may be created via the application ofrecombinant DNA technology, in which changes in the protein structuremay be engineered, based on considerations of the properties of theamino acids being exchanged. Changes designed by man may be introducedthrough the application of site-directed mutagenesis techniques or maybe introduced randomly and screened later for the desired function.

If desired, one also may prepare fusion proteins and peptides, e.g.,where the p16 coding regions are fused with coding regions for otherproteins or peptides and having desired functions, such as forpurification, immunodetection, stabilization or targeting purposes.Furthermore, these fusion proteins or fusion peptides might contain anintracellular targeting sequence that would direct their transport toselected cellular compartments, particularly the nucleus. These fusionproteins or fusion peptides may be expressed from a DNA construct thathas been delivered to animal cells.

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to coding nucleicacid sequences that may, for example, include various non-codingsequences flanking either of the 5′ or 3′ portions of the coding region,such as promoters. Furthermore, affinity or detection moieties, such asdigoxigenin or avidin, may be added to the nucleic acid sequences.

As mentioned above, modification and changes may be made in the primarystructure of p16 (as exemplified by FIG. 1B) and still obtain a moleculehaving like or otherwise desirable characteristics. For example, certainamino acids may be substituted for other amino acids in a proteinstructure without appreciable loss of interactive binding capacity withstructures such as, for example, antigen-binding regions of antibodiesor binding sites on substrate molecules, receptors, or signaltransduction. Since it is the interactive capacity and nature of aprotein that defines that protein's biological functional activity,certain amino acid sequence substitutions can be made in a proteinsequence (or, of course, its underlying DNA coding sequence) andnevertheless obtain a protein with like (agonistic) properties. Equally,the same considerations may be employed to create a protein orpolypeptide with countervailing (e.g., antagonistic) properties. It isthus contemplated by the inventors that various changes may be made inthe sequence of p16 proteins or peptides (or underlying DNA) withoutappreciable loss of their biological utility or activity.

It also is well understood by the skilled artisan that, inherent in thedefinition of a biologically functional equivalent protein or peptide,is the concept that there is a limit to the number of changes that maybe made within a defined portion of the molecule and still result in amolecule with an acceptable level of equivalent biological activity.Biologically functional equivalent peptides are thus defined herein asthose peptides in which certain, not most or all, of the amino acids maybe substituted. In particular, where the N-terminus of the p16 proteinis concerned, it is contemplated that only about 16 or more preferably,about 5 amino acids may be changed within a given peptide. Of course, aplurality of distinct proteins/peptides with different substitutions mayeasily be made and used in accordance with the invention.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. An analysisof the size, shape and type of the amino acid side-chain substituentsreveals that arginine, lysine and histidine are all positively chargedresidues; that alanine, glycine and serine are all a similar size; andthat phenylalanine, tryptophan and tyrosine all have a generally similarshape. Therefore, based upon these considerations, arginine, lysine andhistidine; alanine, glycine and serine; and phenylalanine, tryptophanand tyrosine; are defined herein as biologically functional equivalents.

In making changes, the hydropathic index of amino acids may beconsidered. Each amino acid has been assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics, these are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte & Doolittle, 1982, incorporated herein by reference). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index or score and still retain asimilar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine(−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4).

In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

While the preceding discussion has focused on functionally equivalentpolypeptides arising from amino acid changes, it will be appreciatedthat these changes may be effected by alteration of the encoding DNA,taking into consideration also that the genetic code is degenerate andthat two or more codons may code for the same amino acid.

In addition to the peptidyl compounds described herein, the inventorsalso contemplate that other sterically similar compounds may beformulated to mimic the key portions of the peptide structure. Suchcompounds, which may be termed peptidomimetics, may be used in the samemanner as the peptides of the invention and hence are also functionalequivalents. The generation of a structural functional equivalent may beachieved by the techniques of modelling and chemical design known tothose of skill in the art. It will be understood that all suchsterically similar constructs fall within the scope of the presentinvention.

B. ANTISENSE CONSTRUCTS

In an alternative embodiment, the p16 nucleic acid may encode theantisense version of any of the above full-length or fragmentary nucleicacids. The sense or coding constructs will generally be used in methodsfor inhibiting tumor proliferation where the lack of p16 function is aproblem and replacement of p16 function is desired. However, inembodiments where overexpression of p16 is a problem, such as whereinhibition or suppression of p16 expression is desired, antisensemolecules may be employed.

The term “antisense nucleic acid” is intended to refer to theoligonucleotides complementary to the base sequences of p16-encoding DNAor RNA. Antisense oligonucleotides, when introduced into a target cell,specifically bind to their target nucleic acid and interfere withtranscription, RNA processing, transport, translation and/or stability.Targeting double-stranded (ds) DNA with oligos or oligonucleotides leadsto triple-helix formation; targeting RNA will lead to double-helixformation.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. Antisense RNA constructs, or DNA encoding such antisense RNA's,may be employed to inhibit gene transcription or translation or bothwithin a host cell, either in vitro or in vivo, such as within a hostanimal, including a human subject. Nucleic acid sequences which comprise“complementary nucleotides” are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,that the larger purines will base pair with the smaller pyrimidines toform combinations of guanine paired with cytosine (G:C) and adeninepaired with either thymine (A:T), in the case of DNA, or adenine pairedwith uracil (A:U) in the case of RNA. Inclusion of less common basessuch as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine andothers in hybridizing sequences does not interfere with pairing.

As used herein, the terms “complementary” or “antisense sequences” meannucleic acid sequences that are substantially complementary over theirentire length and have very few base mismatches. For example, nucleicacid sequences of fifteen bases in length may be termed complementarywhen they have a complementary nucleotide at thirteen or fourteenpositions with only a single mismatch. Naturally, nucleic acid sequenceswhich are “completely complementary” will be nucleic acid sequenceswhich are entirely complementary throughout their entire length and haveno base mismatches.

Other sequences with lower degrees of homology also are contemplated.For example, an antisense construct which has limited regions of highhomology, but also contains a non-homologous region (e.g., a ribozyme)could be designed. These molecules, though having less than 50%homology, would bind to target sequences under appropriate conditions.

While all or part of the gene sequence may be employed in the context ofantisense construction, statistically, any sequence of 17 bases longshould occur only once in the human genome and, therefore, suffice tospecify a unique target sequence. Although shorter oligomers are easierto make and increase in vivo accessibility, numerous other factors areinvolved in determining the specificity of hybridization. Both bindingaffinity and sequence specificity of an oligonucleotide to itscomplementary target increases with increasing length. It iscontemplated that oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more base pairs will be used. One can readilydetermine whether a given antisense nucleic acid is effective attargeting of the corresponding host cell gene simply by testing theconstructs in vitro to determine whether the endogenous gene's functionis affected or whether the expression of related genes havingcomplementary sequences is affected.

In certain embodiments, one may wish to employ antisense constructswhich include other elements, for example, those which include C-5propyne pyrimidines. Oligonucleotides which contain C-5 propyneanalogues of uridine and cytidine have been shown to bind RNA with highaffinity and to be potent antisense inhibitors of gene expression(Wagner et al., 1993).

As an alternative to targeted antisense delivery, targeted ribozymes maybe used. The term “ribozyme” is refers to an RNA-based enzyme capable oftargeting and cleaving particular base sequences in p16 DNA and RNA.Ribozymes can either be targeted directly to cells, in the form of RNAoligonucleotides incorporating ribozyme sequences, or introduced intothe cell as an expression construct encoding the desired ribozymal RNA.Ribozymes may be used and applied in much the same way as described forantisense nucleic acids. Ribozyme sequences also may be modified in muchthe same way as described for antisense nucleic acids. For example, onecould incorporate non-Watson-Crick bases, or make mixed RNA/DNAoligonucleotides, or modify the phosphodiester backbone.

C. EXPRESSION CONSTRUCTS

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. Thus, in certainembodiments, expression includes both transcription of a p16 gene andtranslation of a p16 mRNA into a p16 gene product. In other embodiments,expression only includes transcription of the nucleic acid encoding ap16.

In preferred embodiments, the nucleic acid encoding a p16-derivedproduct is under transcriptional control of a promoter. A “promoter”refers to a DNA sequence recognized by the synthetic machinery of thecell, or introduced synthetic machinery, required to initiate thespecific transcription of a gene. The phrase “under transcriptionalcontrol” means that the promoter is in the correct location andorientation in relation to the nucleic acid to control RNA polymeraseinitiation and expression of the gene.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

The particular promoter that is employed to control the expression of anucleic acid encoding a p16 is not believed to be important, so long asit is capable of expressing the nucleic acid in the targeted cell. Thus,where a human cell is targeted, it is preferable to position the nucleicacid coding region adjacent to and under the control of a promoter thatis capable of being expressed in a human cell. Generally speaking, sucha promoter might include either a human or viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter and the Rous sarcoma virus longterminal repeat can be used to obtain high-level expression of p16. Theuse of other viral or mammalian cellular or bacterial phage promoterswhich are well-known in the art to achieve expression of a p16 iscontemplated as well, provided that the levels of expression aresufficient for a given purpose.

By employing a promoter with well-known properties, the level andpattern of expression of a p16 following transfection can be optimized.For example, selection of a promoter which is active specifically inlung cells, such as tyrosinase (melanoma), alpha-fetoprotein and albumin(liver tumors), CC10 (lung tumor) and prostate-specific antigen(prostate tumor) will permit tissue-specific expression of a p16.Further, selection of a promoter that is regulated in response tospecific physiologic signals can permit inducible expression of p16. Forexample, with the nucleic acid encoding p16 being expressed from thehuman PAI-1 promoter, expression is inducible by tumor necrosis factor.Tables 2 and 3 list several elements/promoters which may be employed, inthe context of the present invention, to regulate the expression of p16.This list is not intended to be exhaustive of all the possible elementsinvolved in the promotion of p16 expression but, merely, to be exemplarythereof.

Enhancers were originally detected as genetic elements that increasedtranscription from a promoter located at a distant position on the samemolecule of DNA. This ability to act over a large distance had littleprecedent in classic studies of prokaryotic transcriptional regulation.Subsequent work showed that regions of DNA with enhancer activity areorganized much like promoters. That is, they are composed of manyindividual elements, each of which binds to one or more transcriptionalproteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are often overlapping and contiguous, oftenseeming to have a very similar modular organization.

Below is a list of viral promoters, cellular promoters/enhancers andinducible promoters/enhancers that could be used in combination with thenucleic acid encoding a p16 in an expression construct (Table 2 andTable 3). Additionally any promoter/enhancer combination (as per theEukaryotic Promoter Data Base EPDB) could also be used to driveexpression of a p16. Use of a T3, T7 or SP6 cytoplasmic expressionsystem is another possible embodiment. Eukaryotic cells can supportcytoplasmic transcription from certain bacterial promoters if theappropriate bacterial polymerase is provided, either as part of thedelivery complex or as an additional genetic expression construct.

TABLE 2 ENHANCER Immunoglobulin Heavy Chain Immunoglobulin Light ChainT-Cell Receptor HLA DQ α and DQ β β-Interferon Interleukin-2Interleukin-2 Receptor MHC Class II 5_(a) ^(k) MHC Class II HLA-DRαβ-Actin Muscle Creatine Kinase Prealbumin (Transthyretin) Elastase IMetallothionein Collagenase Albumin Gene α-Fetoprotein τ-Globin β-Globinc-fos c-HA-ras Insulin Neural Cell Adhesion Molecule (NCAM)α_(1-Antitrypsin) H2B (TH2B) Histone Mouse or Type I CollagenGlucose-Regulated Proteins (GRP94 and GRP78) Rat Growth Hormone HumanSerum Amyloid A (SAA) Troponin I (TN I) Platelet-Derived Growth FactorDuchenne Muscular Dystrophy SV40 Polyoma Retroviruses Papilloma VirusHepatitis B Virus Human Immunodeficiency Virus Cytomegalovirus GibbonApe Leukemia Virus

TABLE 3 Element Inducer MT II Phorbol Ester (TFA) Heavy metals MMTV(mouse mammary Glucocorticoids tumor virus) β-Interferon poly(rI)Xpoly(rc) Adenovirus 5 E2 Ela c-jun Phorbol Ester (TPA), H₂O₂ CollagenasePhorbol Ester (TPA) Stromelysin Phorbol Ester (TPA), IL-1 SV40 PhorbolEster (TFA) Murine MX Gene Interferon, Newcastle Disease Virus GRP78Gene A23187 α-2-Macroglobulin IL-6 Vimentin Serum MHC Class I Gene H-2kBInterferon HSP70 Ela, SV40 Large T Antigen Proliferin Phorbol Ester-TPATumor Necrosis Factor FMA Thyroid Stimulating Hormone Thyroid Hormone αGene

In certain embodiments of the invention, the delivery of a nucleic acidin a cell may be identified in vitro or in vivo by including a marker inthe expression construct. The marker would result in an identifiablechange to the transfected cell permitting easy identification ofexpression. Usually the inclusion of a drug selection marker aids incloning and in the selection of transformants. Alternatively, enzymessuch as herpes simplex virus thymidine kinase (tk) (eukaryotic) orchloramphenicol acetyltransferase (CAT) prokaryotic) m ay be employed.Immunologic markers also can be employed. The selectable marker employedis not believed to be important, so long as it is capable of beingexpressed simultaneously with the nucleic acid encoding a p16. Furtherexamples of selectable markers are well known to one of skill in theart.

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the p16transcript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. The inventors have employed the SV40polyadenylation signal in that it was convenient and known to functionwell in the target cells employed. Also contemplated as an element ofthe expression cassette is a terminator. These elements can serve toenhance message levels and to minimize read through from the cassetteinto other sequences.

In preferred embodiments of the invention, the expression constructcomprises a virus or engineered construct derived from a viral genome.The ability of certain viruses to enter cells via receptor-mediatedendocytosis and to integrate into host cell genome and express viralgenes stably and efficiently have made them attractive candidates forthe transfer of foreign genes into mammalian cells (Ridgeway, 1988;Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986).The first viruses used as gene vectors were DNA viruses including thepapovaviruses (simian virus 40, bovine papilloma virus, and polyoma)(Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway,1988; Baichwal and Sugden, 1986). These have a relatively low capacityfor foreign DNA sequences and have a restricted host spectrum.Furthermore, their oncogenic potential and cytopathic effects inpermissive cells raise safety concerns. They can accommodate only up to8 kilobases of foreign genetic material but can be readily introduced ina variety of cell lines and laboratory animals (Nicolas and Rubenstein,1988; Temin, 1986).

(i) Retroviruses

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene, termed Ψ, functions as a signal for packaging of the genomeinto virions. Two long terminal repeat (LTR) sequences are present atthe 5′ and 3′ ends of the viral genome. These contain strong promoterand enhancer sequences and are also required for integration in the hostcell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding a p16is inserted into the viral genome in the place of certain viralsequences to produce a virus that is replication-defective. In order toproduce virions, a packaging cell line containing the gag, pol, and envgenes but without the LTR and Ψ components is constructed (Mann et al.,1983). When a recombinant plasmid containing a human cDNA, together withthe retroviral LTR and Ψ sequences is introduced into this cell line (bycalcium phosphate precipitation for example), the Ψ sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

There are certain limitations to the use of retrovirus vectors in allaspects of the present invention. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the function of flanking genes (Varmus et al., 1981). Anotherconcern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which the intact Ψsequence from the recombinant virus inserts upstream from the gag, pol,env sequence integrated in the host cell genome. However, new packagingcell lines are now available that should greatly decrease the likelihoodof recombination (Markowitz et al., 1988; Hersdorffer et al., 1990).

One limitation to the use of retrovirus vectors in vivo is the limitedability to produce retroviral vector titers greater than 10⁶ infectiousU/mL. Titers 10- to 1,000-fold higher are necessary for many in vivoapplications.

Several properties of the retrovirus have limited its use in lung cancertreatment (Stratford-Perricaudet and Perricaudet, 1991: (i) Infection byretrovirus depends on host cell division. In human cancer, very fewmitotic cells can be found in tumor lesions (Warner and Heston, 1991).(ii) The integration of retrovirus into the host genome may causeadverse effects on target cells, because malignant cells are high ingenetic instability. (iii) Retrovirus infection is often limited by acertain host range. (iv) Retrovirus has been associated with manymalignancies in both mammals and vertebrates. (v) The titer ofretrovirus, in general, is 100- to 1,000-fold lower than that ofadenovirus.

(ii) Adenovirus

Knowledge of the genetic organization of adenovirus, a 36 kB, linear anddouble-stranded DNA virus, allows substitution of a large piece ofadenoviral DNA with foreign sequences up to 7 kB (Grunhaus and Horwitz,1992). In contrast to retrovirus, the infection of adenoviral DNA intohost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in the human.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget-cell range, and high infectivity. Both ends of the viral genomecontain 100-200 base pair (bp) inverted terminal repeats (ITR), whichare cis elements necessary for viral DNA replication and packaging. Theearly (E) and late (L) regions of the genome contain differenttranscription units that are divided by the onset of viral DNAreplication. The E1 region (E1A and E1B) encodes proteins responsiblefor the regulation of transcription of the viral genome and a fewcellular genes. The expression of the E2 region (E2A and E2B) results inthe synthesis of the proteins for viral DNA replication. These proteinsare involved in DNA replication, late gene expression, and host cellshut off (Renan, 1990). The products of the late genes, including themajority of the viral capsid proteins, are expressed only aftersignificant processing of a single primary transcript issued by themajor late promoter (MLP). The MLP (located at 16.8 m.u.) isparticularly efficient during the late phase of infection, and all themRNAs issued from this promoter possess a 5′ tripartite leader (TL)sequence which makes them preferred mRNAs for translation.

In the current system, recombinant adenovirus is generated fromhomologous recombination between shuttle vector and provirus vector. Dueto the possible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure. Use of the YAC system is an alternative approachfor the production of recombinant adenovirus.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham, et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones and Shenk, 1978), the current adenovirus vectors, with the helpof 293 cells, carry foreign DNA in either the E1, the E3 or both regions(Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury, et al.,1987), providing capacity for about 2 extra kB of DNA. Combined with theapproximately 5.5 kB of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kB, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-borne cytotoxicity. Also, the replication deficiency ofthe E1 deleted virus is incomplete. For example, leakage of viral geneexpression has been observed with the currently available adenovirusvectors at high multiplicities of infection (Mulligan, 1993).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in themethod of the present invention. This is because Adenovirus type 5 is ahuman adenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the nucleic acid encodingp16 at the position from which the E1 coding sequences have beenremoved. However, the position of insertion of the p16 coding regionwithin the adenovirus sequences is not critical to the presentinvention. The nucleic acid encoding a p16 transcription unit also maybe inserted in lieu of the deleted E3 region in E3 replacement vectorsas described previously by Karlsson et. al. (1986) or in the E4 regionwhere a helper cell line or helper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10⁹-10¹¹ plaque-forming unit (PFU)/ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal, and therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Experiments in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injection (Herz and Gerard, 1993), andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

(iii) Other Viral Vectors as Expression Constructs

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. recently introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas cotransfected with wild-type virus into an avian hepatoma cell line.Culture media containing high titers of the recombinant virus were usedto infect primary duckling hepatocytes. Stable CAT gene expression wasdetected for at least 24 days after transfection (Chang et al., 1991).

D. METHODS FOR GENE TRANSFER

In order to effect expression of sense or antisense p16 constructs, theexpression construct must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo (see below), as in the treatment ofcertain disease states. As described above, the preferred mechanism fordelivery is via viral infection where the expression construct isencapsidated in an infectious viral particle.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979) andlipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987),gene bombardment using high velocity microprojectiles (Yang et al.,1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,1988). Some of these techniques may be successfully adapted for in vivoor ex vivo use.

Once the expression construct has been delivered into the cell thenucleic acid encoding p16 may be positioned and expressed at differentsites. In certain embodiments, the nucleic acid encoding p16 may bestably integrated into the genome of the cell. This integration may bein the cognate location and orientation via homologous recombination(gene replacement) or it may be integrated in a random, non-specificlocation (gene augmentation). In yet further embodiments, the nucleicacid may be stably maintained in the cell as a separate, episomalsegment of DNA. Such nucleic acid segments or “episomes” encodesequences sufficient to permit maintenance and replication independentof or in synchronization with the host cell cycle. How the expressionconstruct is delivered to a cell and where in the cell the nucleic acidremains is dependent on the type of expression construct employed.

In one embodiment of the invention, the expression construct may simplyconsist of naked recombinant DNA or plasmids. Transfer of the constructmay be performed by any of the methods mentioned above which physicallyor chemically permeabilize the cell membrane. This is particularlyapplicable for transfer in vitro but it may be applied to in vivo use aswell. Dubensky et al. (1984) successfully injected polyomavirus DNA inthe form of CaPO₄ precipitates into liver and spleen of adult andnewborn mice demonstrating active viral replication and acute infection.Benvenisty and Neshif (1986) also demonstrated that directintraperitoneal injection of CaPO₄ precipitated plasmids results inexpression of the transfected genes. It is envisioned that DNA encodinga p16 may also be transferred in a similar manner in vivo and expressp16.

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e., ex vivo treatment. Again, DNA encoding a p16 may be delivered viathis method and still be incorporated by the present invention.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Wong et al. (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells. Nicolau et al. (1987)accomplished successful liposome-mediated gene transfer in rats afterintravenous injection.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

Other expression constructs which can be employed to deliver a nucleicacid encoding a p16 into cells are receptor-mediated delivery vehicles.These take advantage of the selective uptake of macromolecules byreceptor-mediated endocytosis in almost all eukaryotic cells. Because ofthe cell type-specific distribution of various receptors, the deliverycan be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide,a galactose-terminal asialganglioside, incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes.Thus, it is feasible that a nucleic acid encoding a p16 also may bespecifically delivered into a cell type such as lung, epithelial ortumor cells, by any number of receptor-ligand systems with or withoutliposomes. For example, epidermal growth factor (EGF) may be used as thereceptor for mediated delivery of a nucleic acid encoding a p16 in manytumor cells that exhibit upregulation of EGF receptor. Mannose can beused to target the mannose receptor on liver cells. Also, antibodies toCD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma)can similarly be used as targeting moieties.

In certain embodiments, gene transfer may more easily be performed underex vivo conditions. Ex vivo gene therapy refers to the isolation ofcells from an animal, the delivery of a nucleic acid into the cells, invitro, and then the return of the modified cells back into an animal.This may involve the surgical removal of tissue/organs from an animal orthe primary culture of cells and tissues. Anderson et al., U.S. Pat. No.5,399,346, and incorporated herein in its entirety, disclose ex vivotherapeutic methods.

Primary mammalian cell cultures may be prepared in various ways. Inorder for the cells to be kept viable while in vitro and in contact withthe expression construct, it is necessary to ensure that the cellsmaintain contact with the correct ratio of oxygen and carbon dioxide andnutrients but are protected from microbial contamination. Cell culturetechniques are well documented and are disclosed herein by reference(Freshner, 1992).

During in vitro culture conditions the expression construct may thendeliver and express a nucleic acid encoding a p16 into the cells.Finally, the cells may be reintroduced into the original animal, oradministered into a distinct animal, in a pharmaceutically acceptableform by any of the means described below. Thus, providing an ex vivomethod of treating a mammal with a pathologic condition is within thescope of the invention.

E. p16 EXPRESSION CONSTRUCTS IN COMBINATION WITH OTHER THERAPIES

Tumor cell resistance to DNA damaging agents represents a major problemin clinical oncology. One goal of current cancer research is to findways to improve the efficacy of chemo- and radiotherapy by combining itwith gene therapy. For example, the herpes simplex-thymidine kinase(HS-tK) gene, when delivered to brain tumors by a retroviral vectorsystem, successfully induced susceptibility to the antiviral agentganciclovir (Culver, et al., 1992). In the context of the presentinvention, it is contemplated that p16 replacement therapy could be usedsimilarly in conjunction with chemo- or radiotherapeutic intervention.

To kill cells, such as malignant or metastatic cells, using the methodsand compositions of the present invention, one would generally contact a“target” cell with a p16 expression construct and at least one DNAdamaging agent. These compositions would be provided in a combinedamount effective to kill or inhibit proliferation of the cell. Thisprocess may involve contacting the cells with the p16 expressionconstruct and the DNA damaging agent(s) or factor(s) at the same time.This may be achieved by contacting the cell with a single composition orpharmacological formulation that includes both agents, or by contactingthe cell with two distinct compositions or formulations, at the sametime, wherein one composition includes the p16 expression construct andthe other includes the DNA damaging agent.

Alternatively, the p16 treatment may precede or follow the DNA damagingagent treatment by intervals ranging from minutes to weeks. Inembodiments where the DNA damaging factor and p16 expression constructare applied separately to the cell, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the DNA damaging agent and p16 expression constructwould still be able to exert an advantageously combined effect on thecell. In such instances, it is contemplated that one would contact thecell with both agents within about 12-24 hours of each other and, morepreferably, within about 6-12 hours of each other, with a delay time ofonly about 12 hours being most preferred. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

It also is conceivable that more than one administration of either p16or the DNA damaging agent will be desired. Various combinations may beemployed, where p16 is “A” and the DNA damaging agent is “B”:

A/B/A    B/A/B B/B/A A/A/B    B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/AB/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B

To achieve cell killing, both agents are delivered to a cell in acombined amount effective to kill the cell.

DNA damaging agents or factors are defined herein as any chemicalcompound or treatment method that induces DNA damage when applied to acell. Such agents and factors include radiation and waves that induceDNA damage such as, γ-irradiation, X-rays, UV-irradiation, microwaves,electronic emissions, and the like. A variety of chemical compounds,also described as “chemotherapeutic agents”, function to induce DNAdamage, all of which are intended to be of use in the combined treatmentmethods disclosed herein. Chemotherapeutic agents contemplated to be ofuse, include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide (VP-16),camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and evenhydrogen peroxide. The invention also encompasses the use of acombination of one or more DNA damaging agents, whether radiation-basedor actual compounds, such as the use of X-rays with cisplatin or the useof cisplatin with etoposide. In certain embodiments, the use ofcisplatin in combination with a p16 expression construct is particularlypreferred as this compound.

In treating cancer according to the invention, one would contact thetumor cells with a DNA damaging agent in addition to the p16 expressionconstruct. This may be achieved by irradiating the localized tumor sitewith DNA damaging radiation such as X-rays, UV-light, γ-rays or evenmicrowaves. Alternatively, the tumor cells may be contacted with the DNAdamaging agent by administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising a DNAdamaging compound such as, adriamycin, 5-fluorouracil, etoposide,camptothecin, actinomycin-D, mitomycin C, or more preferably, cisplatin.The DNA damaging agent may be prepared and used as a combinedtherapeutic composition, or kit, by combining it with a p16 expressionconstruct, as described above.

Agents that directly cross-link nucleic acids, specifically DNA, areenvisaged and are shown herein, to eventuate DNA damage leading to asynergistic antineoplastic combination. Agents such as cisplatin, andother DNA alkylating may be used. Cisplatin has been widely used totreat cancer, with efficacious doses used in clinical applications of 20mg/m² for 5 days every three weeks for a total of three courses.Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. As such a number ofnucleic acid precursors have been developed. Particularly useful areagents that have undergone extensive testing and are readily available.As such, agents such as 5-fluorouracil (5-FU), are preferentially usedby neoplastic tissue, making this agent particularly useful fortargeting to neoplastic cells. Although quite toxic, 5-FU, is applicablein a wide range of carriers, including topical, however intravenousadministration with doses ranging from 3 to 15 mg/kg/day being commonlyused.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageDNA, on the precursors of DNA, the replication and repair of DNA, andthe assembly and maintenance of chromosomes. Dosage ranges for X-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

The inventors propose that the regional delivery of p16 expressionconstructs to patients with p16-linked cancers will be a very efficientmethod for delivering a therapeutically effective gene to counteract theclinical disease. Similarly, the chemo- or radiotherapy may be directedto a particular, affected region of the subjects body. Alternatively,systemic delivery of p16 expression construct or the DNA damaging agentmay be appropriate in certain circumstances, for example, whereextensive metastasis has occurred.

In addition to combining p16-targeted therapies with chemo- andradiotherapies, it also is contemplated that combination with other genetherapies will be advantageous. For example, targeting of p16 and p53mutations at the same time may produce an improved anti-cancertreatment. Any other tumor-related gene conceivably can be targeted inthis manner, for example, p21, Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I,MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf, erb, src, fms, jun,trk, ret, gsp, hst, bcl and abl.

F. NUCLEIC ACID ENCODING p16 OR p16 AS MARKERS

In certain embodiments, a nucleic acid encoding a p16 or p16 peptide maybe employed for diagnostic purposes. The absence or reduced/increasedlevel of p16 nucleic acid encoding a p16 may be indicative of a diseasestate such as cancer. Thus, the present invention also includes usingnucleic acid encoding a p16 or p16 as a marker. There are numerousmethods, well known to one of skill in the art, that may be employed indetecting a nucleic acid encoding p16 or a p16. Two common methods fordetecting nucleic acids encoding a p16 are Southern and Northernanalyses and variations thereof. The level of p16 message can be used asa marker to indicate tumorigenicity.

An alternative approach would be to detect p16 with immunoassays usingantibodies that bind to p16 in Western Blotting and FACS analysis arealso described in Example I. Both techniques were employed to detect p16expression on the cell surface. It will be readily appreciated thatdetection is not limited to the above techniques, and that there arenumerous other methods which may be encompassed by the presentinvention.

Other, preferred immunoassays are the various types of enzyme-linkedimmunosorbent assays (ELISA's) and radioimmunoassays (RIA's) known inthe art. Immunohistochemical detection using tissue sections also isparticularly useful.

In ELISA's, an anti-p16 antibody (such as Ab669, as disclosed herein) isimmobilized onto a selected surface exhibiting protein affinity, such asa well in a polystyrene microtiter plate. Then, a test compositioncontaining the cells or cellular material, such as a clinical sample, isadded to the wells. After binding and washing to remove non-specificallybound immunocomplexes, the bound p16 may be detected. Detection isgenerally achieved by the addition of another anti-p16 antibody that islinked to a detectable label. This type of ELISA is a simple “sandwichELISA”. Detection may also be achieved by the addition of a secondanti-p16 antibody, followed by the addition of a third antibody that hasbinding affinity for the second anti-p16 antibody, with the thirdantibody being linked to a detectable label.

In another exemplary ELISA, the samples containing the cellular materialto be tested for the level of p16, are immobilized onto the well surfaceand then contacted with the anti-p16 antibodies. After binding andappropriate washing, the bound immunocomplexes are detected. Where theinitial anti-p16 antibodies are linked to a detectable label, theimmunocomplexes may be detected directly. Again, the immunocomplexes maybe detected using a second antibody that has binding affinity for thefirst anti-p16 antibody, with the second antibody being linked to adetectable label.

Competition ELISA's also are possible in which test samples compete forbinding with known amounts of labeled p16 antigens or antibodies. Theamount of reactive species in the unknown sample is determined by mixingthe sample with the known labeled species before or during incubationwith coated wells. The presence of reactive species in the sample actsto reduce the amount of labeled species available for binding to thewell and thus reduces the ultimate signal.

Irrespective of the format employed, ELISA's have certain features incommon, such as coating, incubating or binding, washing to removenon-specifically bound species, and detecting the bound immunocomplexes.These are described as follows:

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein, gelatin, and solutions of milk powder. The blocking solutionsalso usually contain the detergent Tween-20, which greatly helps toreduce non-specific binding. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISA's, it is more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding ofthe p16 or anti-p16 antibody to the well, coating with a non-reactivematerial to reduce background, and washing to remove unbound material,the immobilizing surface is contacted with the clinical or biologicalsample to be tested under conditions effective to allow immunocomplex(antigen/antibody) formation. Detection of the immunocomplex thenrequires a labeled secondary binding ligand or antibody, or a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or third binding ligand.

“Under conditions effective to allow immunocomplex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and antibodies with solutions such as BSA, bovine gammaglobulin (BGG) and phosphate buffered saline (PBS)/Tween-20. These addedagents also tend to assist in the reduction of nonspecific background.

The suitable conditions also mean that the incubation is at atemperature and for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours, attemperatures preferably on the order of 25° to 27° C., or may beovernight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. Washing often includeswashing with a solution of PBS/Tween-20, or borate buffer. Following theformation of specific immunocomplexes between the test sample and theoriginally bound material, and subsequent washing, the occurrence ofeven minute amounts of immunocomplexes may be determined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. Preferably, this will be an enzymethat will generate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact andincubate the first or second immunocomplex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immunocomplex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS/Tween-20).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS)and H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generation, e.g.,using a visible spectra spectrophotometer.

F. PHARMACEUTICAL COMPOSITIONS AND ROUTES OF ADMINISTRATION

Where clinical application of an expression construct comprising anucleic acid encoding p16 is contemplated, it will be necessary toprepare the complex as a pharmaceutical composition appropriate for theintended application. Generally this will entail preparing apharmaceutical composition that is essentially free of pyrogens, as wellas any other impurities that could be harmful to humans or animals. Onealso will generally desire to employ appropriate salts and buffers torender the complex stable and allow for complex uptake by target cells.

Aqueous compositions of the present invention comprise an effectiveamount of the expression construct and nucleic acid, dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium.Such compositions can also be referred to as inocula. The phrases“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions also can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The expression constructs and delivery vehicles of the present inventionmay include classic pharmaceutical preparations. Administration oftherapeutic compositions according to the present invention will be viaany common route so long as the target tissue is available via thatroute. This includes oral, nasal, buccal, rectal, vaginal or topical.Topical administration would be particularly advantageous for treatmentof skin cancers, to prevent chemotherapy-induced alopecia or otherdermal hyperproliferative disorder. Alternatively, administration willbe by orthotopic, intradermal subcutaneous, intramuscular,intraperitoneal or intravenous injection. Such compositions wouldnormally be administered as pharmaceutically acceptable compositionsthat include physiologically acceptable carriers, buffers or otherexcipients.

The therapeutic compositions of the present invention are advantageouslyadministered in the form of injectable compositions either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. Thesepreparations also may be emulsified. A typical composition for suchpurpose comprises a pharmaceutically acceptable carrier. For instance,the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg ofhuman serum albumin per milliliter of phosphate buffered saline. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oil and injectable organic esters such asethyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, saline solutions, parenteral vehicles such as sodiumchloride, Ringer's dextrose, etc. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial agents,anti-oxidants, chelating agents and inert gases. The pH and exactconcentration of the various components the pharmaceutical compositionare adjusted according to well known parameters.

Additional formulations are suitable for oral administration. Oralformulations include such typical excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders. When the route istopical, the form may be a cream, ointment, salve or spray.

An effective amount of the therapeutic agent is determined based on theintended goal, for example (i) inhibition of tumor cell proliferation or(ii) elimination of tumor cells. The term “unit dose” refers tophysically discrete units suitable for use in a subject, each unitcontaining a predetermined-quantity of the therapeutic compositioncalculated to produce the desired responses, discussed above, inassociation with its administration, i.e., the appropriate route andtreatment regimen. The quantity to be administered, both according tonumber of treatments and unit dose, depends on the subject to betreated, the state of the subject and the protection desired. Preciseamounts of the therapeutic composition also depend on the judgment ofthe practitioner and are peculiar to each individual.

G. KITS

All the essential materials and reagents required for inhibiting tumorcell proliferation, transforming cells or detecting cancer cells, may beassembled together in a kit. This generally will comprise selectedexpression constructs. Also included may be various media forreplication of the expression constructs and host cells for suchreplication. Such kits will comprise distinct containers for eachindividual reagent.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution preferably is an aqueous solution, with asterile aqueous solution being particularly preferred. For in vivo use,the expression construct may be formulated into a pharmaceuticallyacceptable syringeable composition. In this case, the container meansmay itself be an inhalent, syringe, pipette, eye dropper, or other suchlike apparatus, from which the formulation may be applied to an infectedarea of the body, such as the lungs, injected into an animal, or evenapplied to and mixed with the other components of the kit.

The components of the kit may also be provided in dried or lyophilizedforms. When reagents or components are provided as a dried form,reconstitution generally is by the addition of a suitable solvent. It isenvisioned that the solvent also may be provided in another containermeans.

The kits of the present invention also will typically include a meansfor containing the vials in close confinement for commercial sale suchas, e.g., injection or blow-molded plastic containers into which thedesired vials are retained.

Irrespective of the number or type of containers, the kits of theinvention also may comprise, or be packaged with, an instrument forassisting with the injection/administration or placement of the ultimatecomplex composition within the body of an animal. Such an instrument maybe an inhalent, syringe, pipette, forceps, measured spoon, eye dropperor any such medically approved delivery vehicle.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLES A. MATERIALS AND METHODS

Cell Lines. Cell line 293 was maintained in Eagle's modified essentialmedium supplemented with 10% heat-inactivated horse serum. Human NSCLCcell lines H226Br, H322 were grown in RPMI medium containing 5% fetalbovine serum. Human normal breast cell line HBL100 was grown in F12medium supplemented with 10% fetal bovine serum.

p16^(INK4) cDNA Subcloning. The original p16^(INK4) cDNA was amplifiedfrom the total RNA of normal human lymphocytes by RT-PCR™ using theprimers 5′-ATGGAGCCTTCGGC TGACTGG-3′ (SEQ ID NO:3) and5′-CCTGTAGGACCTTCGGTGACT-3′ (SEQ ID NO:4). The PCR™ product wassubcloned in pCR™ vector (Invitrogen, San Diego, Calif.) and verified bydouble-stranded DNA sequencing. Because of a correction of thep16^(INK4) cDNA sequence in GenBank, an additional sequence of 42 basepairs was added later to the 5′ end of the cloned p16^(INK4) cDNA by twoPCR™ steps. The first PCR™ step used primer A (5′-GATCCGGCGGCGGGGAGCAGCATGGAGCCTTC GGCTGACTGG-3′; SEQ ID NO:5); and primer C(5′-GCCTCTCTGGTTCTTTCA-3′; SEQ ID NO:6). The second PCR™ step usedprimer B (5′-CGGGCGGGGAGCAGCATGGAGCCGG CGGCGGGGAGC-3′; SEQ ID NO:7) andprimer C. The final wild-type p16^(INK4) cDNA sequence in the pCR™vector pCR-p-16) was again verified by double-stranded DNA sequencing.

pAd-p16 Construction. The shuttle vector pEC53 (Zhang et al., 1994) wasdigested by restriction enzymes HinD III AND Hpa I. The vector backbonewas separated from p53 cDNA by running the digested DNA through 1%agarose gel and was purified from the gel. p16^(INK4) cDNA was excisedfrom pCR-p16 and ligated to the purified shuttle vector backbone. Thefinal product, pAd-p16, carries the p16^(INK4) expression cassette whichcontains human CMV promoter (Boshart et al., 1985) wild-type p16^(INK4)cDNA, and SV40 early polyadenylation signal.

Generation of Recombinant p16^(INK4) Adenovirus. The recombinantAd5CMV-lacZ adenovirus DNA was digested with restriction enzymes XbaIand Clal and the 32 kb partial adenovirus DNA fragment was purified in a0.3% agarose gel. This DNA fragment and pAd-p16 plasmid DNA werecotransfected into 293 cells by CaPO₄-mediated transfection. Thetransfected cells were maintained in medium until the onset of thecytopathic effect. The newly generated p16^(INK4) recombinant adenovirus(Ad-p16) was identified by PCR™ analysis of the DNA samples preparedfrom the cell culture supernatant. The recombinant adenovirus Ad5CMV-lacZ which carries the lacZ gene of E. coli, has a structure similarto that of Ad-p16 and was used as a control in these studies.

Viral Stocks, Titers, and Infection. Individual clones of the Ad-p16 andAd5CMV-lacZ viruses were obtained by plaque-purification and werepropagated in 293 cells according to the method of Graham and Prevec(1991). The viral titers were determined by plaque assays. The celllines were infected by addition of the viral solutions to cellmonolayers and incubation at room temperature for 30 min with briefagitation every 5 min. This was followed by the addition of culturemedium and return of the infected cells to the 37° C. incubator.

Tumorigenicity Assays. H460 cells were infected with Ad-p16 orAd5CMV-lacZ at an MOI of 50 PFU/cell. An equal number of cells weretreated with medium only as a mock infection. Twenty-four hours afterinfection, the treated cells were harvested and rinsed twice with PBS.For each treatment, 5×10⁶ cells in a volume of 0.1 ml of PBS wereinjected subcutaneously into the dorsal flank of BALB/c nu/nu mice(Harlan Sprague-Dawley Co., Houston, Tex.). The treated mice wereexamined weekly after injection. Tumor generation was evaluated at theend of a 3-week period. Tumor volume was calculated by assuming aspherical shape with the average tumor diameter calculated as the squareroot of the product of orthogonal diameters.

B. RESULTS

Generation of the Ad-p16 Recombinant Virus. One of the advantages ofadenovirus as a gene transfer vector is that it has high infectivity ina wide range of host cells (Berkner, 1988). An adenovirus-derivedshuttle vector for human cancer gene therapy, pEC53, was constructedpreviously (Zhang et al., 1994). A recombinant virus derived from thisvector, Ad5CMV-p53, has an infectivity of 97% to 100% in several lungcancer cell lines (Zhang et al., 1994), including H460, H322, andH226Br. In this study, the p53 gene in pEC53 was replaced by thefull-length p16^(INK4) cDNA. The final product, pAd-p16, carries thep16^(INK4) gene expression cassette which contains a human CMV promoter(Boshart et al., 1985), wild-type p16^(INK4) cDNA, and a SV40 earlypolyadenylation signal. A 32 kB partial fragment of adenovirus DNAgenerated by Xba I digestion of the DNA of a recombinant virus,Ad5CMV-lacZ, was cotransfected with pAd-p16 plasmid into 293 cells forhomologous recombination. The recombinant viral product, Ad-p16, has agenomic structure similar to that of Ad5CMV-lacZ except that the lacZgene is replaced by the p16^(INK4) gene. Since the E1 regions of therecombinant adenoviruses are substituted by the p16^(INK4) gene or thelacZ gene expression cassette, they can be propagated only in 293 cellsthat complement the E1 deletion. Ad5CMV-lacZ was used as a viral controlfor Ad-p16.

Expression of Exogenous p16^(INK4) Protein in Human Lung Cancer Cells.Three human NSCLC cell lines were chosen for this study: H460, H322 andH226Br. H460 carries homozygous p16^(INK4) gene deletions (Kamb et al.,1994), but the p53 gene (Takahashi et al., 1989) and the Rb gene(Harbour et al., 1988) are wild-type. H322 carries a homozygousp16^(INK4) gene deletion (Okamoto et al., 1994), a wild-type Rb gene(Harbour et al., 1988), and a homozygous p53 mutation at codon 248(Mitsudomi et al., 1992). H226Br carries the p16^(INK4) gene detected bysouthern blot and PCR™ analysis which has not been sequenced, but doesnot express p16^(INK4) at the protein level. It carries a homozygous p53mutation at codon 254 (Fujiwara et al., 1994) and expresses wild-type Rbprotein (Harbour et al., 1988). A cultured normal human breastepithelial cell line, HBL100, expresses wild-type p16^(INK4), wild-typep53 and wild-type RB gene and was used in this study as a normal cellline control. Only cell line HBL100 expressed p16^(INK4) protein beforeviral infection. To obtain a high level of expression of exogenousp16^(INK4) protein, the human CMV promoter (Boshart et al., 1985) wasused to drive the expression of the p16^(INK4) gene. High levels ofexogenous p16^(INK4) protein expression were achieved in the H460, H322and H226Br cells after infection with Ad-p16. The level of p16^(INK4)protein in HBL100 was much higher after Ad-p16 infections, indicatingexogenous p16^(INK4) protein expression in this cell line. Subsequently,the effect(s) of this introduced p16^(INK4) protein on the tumor celllines were examined in the following assays.

Effect of p16^(INK4) Protein on Lung Cancer Cell Growth. The NSCLC celllines H460, H322 and H226Br and the normal breast cell line HBL100 wereinfected with Ad-p16 or Ad5CMV-lacZ at 50 PFU/cell. Triplicate sets ofthe infected and mock-infected cells were counted every day for 6 days,and the mean cell number for each day was calculated. As shown in FIG.2, growth rates of the Ad-p16-infected H460, H322 and H226Br cells wereinhibited by 94%, 85%, and 97%, respectively, compared with that of theAd5CMV-lacZ-infected cells. However, the growth rate of HBL100 infectedwith Ad-p16 was inhibited by less than 3% when compared with that of theAd5CMV-lacZ-infected HBL100 cells. This suggested that introduction ofthe p16^(INK4) gene into these cell lines could specifically suppresscell proliferation by restoring p16^(INK4) expression. The growth ratesof the Ad5CMV-lacZ-treated cells were lower than those of themock-infected cells for all the cell lines, indicating cytotoxicitycaused by expressed viral proteins and the lacZ gene. This virus-relatedcytotoxicity was increased when a higher MOI was used. The cell lineshad differing sensitivities to this effect.

Cell-Cycle Arrest Mediated by Ad-p16. It is known that the p16^(INK4)protein can inhibit the activity of CDK4 and CDK6, thereby blocking theentry of the proliferating cells from G₁ phase to S phase (Serrano etal., 1993; Serrano et al., 1995). To examine the mechanism of the growthrate inhibition mediated by Ad-p16, H460, H322, H226Br and HBL100 cellswere infected as described in the growth rate assay and harvested 24hours after infection for cell cycle analysis by flow cytometry. Asshown in Table 4, Ad-p16-mediated expression of the p16^(INK4) proteinsignificantly increased the numbers of cells in G₁ phase and decreasedthe number of cells in S and (G₂+M) phases in the p16^(INK4) deletedtumor cell lines, suggesting the induction of G₁ arrest. In contrast, noG₁ arrest was observed in the p16^(INK4) protein-positive normal breastcell line, HBL100. These results suggest that the p16^(INK4) proteinsuppresses the growth of the tumor cells by mediating G₁ arrest in celllines that do not express p16^(INK4).

TABLE 4 Flow Cytometry Analysis of Cell-Cycle Effects of p16^(INK4)Mediated by Ad-p16 cell types & Percent of Cells in infected virusesG₀/G₁ S G₂/M HBL100/Ad-p16 36 42 22 HBL100/Ad5CMV-lacZ 31 44 25HBL100/medium 31 42 27 H460/Ad-p16 88 9 3 H460/Ad5CMV-lacZ 41 32 27H460/medium 39 35 26 H322/Ad-p16 80 6 14 H322/Ad5CMV-lacz 30 36 34H322/medium 30 36 34 H226Br/Ad-p16 80 11 9 H226Br/Ad5CMV-lacZ 20 53 27H226Br/medium 26 44 30 Values are shown for a representative assay.Cells were infected the same way as in the cell growth ratedetermination assay. 24 hours after infection, cells were treated with1% Tween-20 and stained with propidium iodide and then analyzed by flowcytometry for DNA synthesis and cell cycle status. Flow cytometric assaywas performed with a FACScan (Becton Dickinson, San Jose, CA) equippedwith an air-cooled 15-mW 488 nm argon laser. Red fluorescence wasmeasured through a long pass # filter with cut-off wavelength at 650 nm.

Inhibition of Tumorigenicity Mediated by Ad-p16. To determine whetherthe Ad-p16 virus can inhibit tumorigenicity of human NSCLC cells, BALB/cnu/nu mice were injected subcutaneously with H460 cells to induce tumorformation. Each mouse received one injection of 5×10⁶ cells that hadbeen infected with either Ad-p16 or Ad5CMV-lacZ at 50 PFU/cell for 24hours. H460 cells treated with medium alone were used as mock-infectedcontrols. Each treatment was given to four mice. The mice were observed,and when tumors appeared they were measured for a 3-week period. Twoindependent studies were done to confirm reproducibility and the datafrom both studies are summarized in Table 5. Ad-p16 treated cellssignificantly suppressed tumor growth in vivo. 100% of the mice thatreceived medium treated cells and 87.5% of the mice that receivedAd5/CMV-lacZ treated cells developed tumors. On the other hand, only 50%mice in both studies that received Ad-p16 treated cells developed tumorsand the mean volume was only 11% of that in Ad5CMV-lacZ virus treatedmice and 6% of that in the medium treated mice (p<0.001 by two sidedstudent's T test). Thus, the tumorigenicity of the lung cancer cells wasinhibited by prior treatment with Ad-p16, indicating that the p16^(INK4)protein may have therapeutic efficacy.

TABLE 5 Effect of p16^(INK4) on Tumorigenicity of H460 Cell Line in NudeMice No. of tumors Mean Volume Treatment No. of mice (%) (mm³ ± SD)Experiment 1 Medium 4/4 (100%) 1047.3 ± 104.7  Ad5/CMV-LacZ 4/4 (100%)740.5 ± 205.5 Ad-p16 0/4 (0%) — Experiment 2 Medium 4/4 (100%) 1158.6 ±200.2  Ad5/CMV-LacZ 3/4 (75%) 658.3 ± 144.3 Ad-p16 4/4 (100%) 71.2 ±19.6 The H460 cells were infected with viruses at a MOI of 50 PFU/cellfor 24 hours and injected subcutaneously at 5 × 10⁶ cells/mouse. Tumorsizes were determined at the end of a three-week period.

The therapeutic potential for Ad-p16 was examined in a BALB/c nu/numouse model. Subcutaneous tumor nodules arose 20 days after injectingmice subcutaneously with 5×10⁶ H460 cells. The resulting tumors weredirectly injected with Ad-p16 (10¹⁰ PFU/tumor), Ad5CMV-lacZ (1010PFU/tumor) or PBS. As shown in FIG. 3, 18 days after injection theaverage volume of Ad-p16-treated tumors was only 49% of that of thecontrol virus-treated tumors and 34% of that of the tumors injected withPBS (p<0.001 by two-sided Student's t test).

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7 987 base pairs nucleic acid single linear other nucleic acid /desc =“DNA” not provided 1 CGGAGAGGGG GAGAACAGAC AACGGGCGGC GGGGAGCAGCATGGAGCCGG CGGCGGGGAG 60 CAGCATGGAG CCTTCGGCTG ACTGGCTGGC CACGGCCGCGGCCCGGGGTC GGGTAGAGGA 120 GGTGCGGGCG CTGCTGGAGG CGGGGGCGCT GCCCAACGCACCGAATAGTT ACGGTCGGAG 180 GCCGATCCAG GTCATGATGA TGGGCAGCGC CCGAGTGGCGGAGCTGCTGC TGCTCCACGG 240 CGCGGAGCCC AACTGCGCCG ACCCCGCCAC TCTCACCCGACCCGTGCACG ACGCTGCCCG 300 GGAGGGCTTC CTGGACACGC TGGTGGTGCT GCACCGGGCCGGGGCGCGGC TGGACGTGCG 360 CGATGCCTGG GGCCGTCTGC CCGTGGACCT GGCTGAGGAGCTGGGCCATC GCGATGTCGC 420 ACGGTACCTG CGCGCGGCTG CGGGGGGCAC CAGAGGCAGTAACCATGCCC GCATAGATGC 480 CGCGGAAGGT CCCTCAGACA TCCCCGATTG AAAGAACCAGAGAGGCTCTG AGAAACCTCG 540 GGAAACTTAG ATCATCAGTC ACCGAAGGTC CTACAGGGCCACAACTGCCC CCGCCACAAC 600 CCACCCCGCT TTCGTAGTTT TCATTTAGAA AATAGAGCTTTTAAAAATGT CCTGCCTTTT 660 AACGTAGATA TAAGCCTTCC CCCACTACCG TAAATGTCCATTTATATCAT TTTTTATATA 720 TTCTTATAAA AATGTAAAAA AGAAAAACAC CGCTTCTGCCTTTTCACTGT GTTGGAGTTT 780 TCTGGAGTGA GCACTCACGC CCTAAGCGCA CATTCATGTGGGCATTTCTT GCGAGCCTCG 840 CAGCCTCCGG AAGCTGTCGA CTTCATGACA AGCATTTTGTGAACTAGGGA AGCTCAGGGG 900 GGTTACTGGC TTCTCTTGAG TCACACTGCT AGCAAATGGCAGAACCAAAG CTCAAATAAA 960 AATAAAATAA TTTTCATTCA TTCACTC 987 156 aminoacids amino acid single linear protein not provided 2 Met Glu Pro AlaAla Gly Ser Ser Met Glu Pro Ser Ala Asp Trp Leu 1 5 10 15 Ala Thr AlaAla Ala Arg Gly Arg Val Glu Glu Val Arg Ala Leu Leu 20 25 30 Glu Ala GlyAla Leu Pro Asn Ala Pro Asn Ser Tyr Gly Arg Arg Pro 35 40 45 Ile Gln ValMet Met Met Gly Ser Ala Arg Val Ala Glu Leu Leu Leu 50 55 60 Leu His GlyAla Glu Pro Asn Cys Ala Asp Pro Ala Thr Leu Thr Arg 65 70 75 80 Pro ValHis Asp Ala Ala Arg Glu Gly Phe Leu Asp Thr Leu Val Val 85 90 95 Leu HisArg Ala Gly Ala Arg Leu Asp Val Arg Asp Ala Trp Gly Arg 100 105 110 LeuPro Val Asp Leu Ala Glu Glu Leu Gly His Arg Asp Val Ala Arg 115 120 125Tyr Leu Arg Ala Ala Ala Gly Gly Thr Arg Gly Ser Asn His Ala Arg 130 135140 Ile Asp Ala Ala Glu Gly Pro Ser Asp Ile Pro Asp 145 150 155 21 basepairs nucleic acid single linear other nucleic acid /desc = “DNA” notprovided 3 ATGGAGCCTT CGGCTGACTG G 21 21 base pairs nucleic acid singlelinear other nucleic acid /desc = “DNA” not provided 4 CCTGTAGGACCTTCGGTGAC T 21 42 base pairs nucleic acid single linear other nucleicacid /desc = “DNA” not provided 5 GATCCGGCGG CGGGGAGCAG CATGGAGCCTTCGGCTGACT GG 42 18 base pairs nucleic acid single linear other nucleicacid /desc = “DNA” not provided 6 GCCTCTCTGG TTCTTTCA 18 36 base pairsnucleic acid single linear other nucleic acid /desc = “DNA” not provided7 CGGGCGGGGA GCAGCATGGA GCCGGCGGCG GGGAGC 36

What is claimed is:
 1. A method of inhibiting tumor growth in a mammalcomprising: (i) providing a pharmaceutical composition comprising (a) anadenoviral vector comprising a promoter functional in eukaryotic cellsand a nucleic acid encoding a p16, said nucleic acid being under thecontrol of said promoter and positioned in a sense orientation to saidpromoter, and (b) a pharmaceutically acceptable buffer, solvent ordiluent; and (ii) administering said pharmaceutical composition to saidmammal by direct intratumoral administration.
 2. The method according toclaim 1, wherein said mammal is a human.
 3. The method according toclaim 1, wherein said tumor is a lung cancer tumor, a bladder cancertumor, an esophageal cancer tumor, a pancreatic cancer tumor, a head andneck cancer tumor, a glioma, or a melanoma.
 4. The method according toclaim 1, wherein said adenoviral vector further comprises apolyadenylation signal 3′ to said p16 encoding nucleic acid.
 5. Themethod according to claim 1, wherein said adenoviral vector is areplication deficient adenovirus.
 6. The method according to claim 5,wherein said adenoviral vector lacks at least a portion of the E1region.
 7. The method according to claim 6, wherein said adenoviralvector lacks the entire E1 region.
 8. The method according to claim 1,wherein said direct intratumoral administration comprises three doses ofadenoviral vector totalling 10¹⁰ PFU.
 9. The method according to claim1, wherein said promoter is selected from the group consisting of CMVIE, SV40 early, RSV LTR, tyrosinase, alpha-fetoprotein, albumin, PSA andPAI-1.
 10. The method according to claim 1, wherein said promoter is CMVIE.
 11. The method according to claim 1, wherein said nucleic acidencoding p16 is a cDNA.
 12. The method according to claim 1, whereinsaid pharmaceutical composition comprises adenoviral vector at 10⁹ to10¹¹ PFU/ml.
 13. The method according to claim 1, wherein saidadministration comprises injection.